Despite significant limitations, opioids remain a mainstay for the treatment of severe acute and chronic pain. Two clinically significant limitations that accompany the long-term therapeutic use of opioids are: (1) The development of tolerance (requiring escalating doses of opioid to maintain the desired therapeutic benefit or leading to diminished benefit with constant dose) and (2) Physical dependence (withdrawal symptoms on cessation of opioid use, which are very unpleasant, and seeking their avoidance may increase the risk for opioid addiction). Currently, there are no practical approaches for decreasing opioid tolerance or suppressing the development of physical dependence. However, in exploring potential interactions between CB2 cannabinoid receptor (CB2R) and mu opioid receptor (MOR) signaling, we made the exciting discovery that certain CB2 agonists effectively prevented the development of tolerance to opioid-induced anti-allodynic efficacy in a murine neuropathic pain model, while also blunting the physical dependence that accompanies chronic morphine exposure. These findings, if they can be translated to humans, hold the promise to significantly improve the clinical use of opioids. On the path to translating these findings, we will complete three specific aims to better understand how CB2 and mu opioid receptor agonists interact to suppress opioid tolerance and dependence: Aim 1: Identify the CB2-expressing cells engaged by LY28282360 to prevent opioid tolerance. We will delineate the cell types responsible for the ability of LY2828360 to block development of morphine tolerance and identify site of action using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 2: Define the conditions under which CB2 agonists suppress opioid-induced physical dependence. We will delineate the cell types responsible for the effects of LY2828360 on opioid dependence, as measured using naloxone precipitated opioid withdrawal, using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 3: Mechanistic characterization of CB2R/MOR interaction. We will characterize CB2 ligands for their G protein/arrestin signaling bias as well as their kinetics of G protein activation. We will also determine if the slow activation of G protein signaling by LY2828360 and related CB2 agonists is due to the kinetics of receptor binding. Finally, if the results of Aims 1 or 2 suggest that MOR and CB2R are interacting in the same cell, we will characterize the differences in CB2R/MOR crosstalk between slowly and rapidly signaling CB2 agonists. Completion of these aims will fully characterize interactions between CB2 and opioid receptors in preclinical and cell-based models. These studies will help define the clinical settings where CB2 agonists may be useful in countering two major limitations on the use of opioids in treating chronic pain.